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CONCLUSIONS AND PERSPECTIVES
In this work we have sought to cover as widely as possible the diverse aspects of enzymology. While it is clear that the fundamental aspects of thermodynamics, enzyme kinetics and the physico-chemical principles of enzymatic catalysis remain unchanged, the data concerning the structure-function relationships of particular enzyme systems are constantly evolving. This is due to the rapid growth in struc-tural knowledge which has resulted from technological progress. It suffices to note that in 1965, the three-dimensional structure of three proteins were solved by X-ray diffraction. These were haemoglobin, myoglobin and lysozyme. In 1970, the three-dimensional structures of 11 proteins were known. In March 2008, nearly 50 000 structures were available in the Protein Data Bank. The figure below illustrates this progress of structural knowledge. The number of structural motifs and protein superfamilies remains more limited.
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Annual deposition of structuresCumulative total
Evolution of the number of protein structures deposited in the Protein Data Bank
Today, we have particularly powerful tools at our disposal, such as synchrotron ra-diation sources, high-field nuclear magnetic resonance spectrometers and powerful
J. Yon-Kahn, G. Hervé, Molecular and Cellular Enzymology, DOI 10.1007/978-3-642-01228-0, © Springer-Verlag Berlin Heidelberg 2010
MOLECULAR AND CELLULAR ENZYMOLOGY
computers which permit the resolution of protein structures and multi-molecular structures. Structural knowledge allied to genetic engineering methods and to pro-tein chemistry enables investigation in increasing detail of the active and regulatory sites of enzymes. Even if each day new data appear, the systems that we have chosen to present serve well as useful paradigms, as much in relation to investiga-tive strategies as to the mechanisms of action brought about by these systems.
Although structural knowledge is necessary for understanding the mechanisms involved in the functioning of enzymes and complex enzyme systems, it is, how-ever, insufficient. The basis of their functioning even lies in the structural dynamics of enzyme molecules. Indeed, proteins are not rigid objects; their structure fluctu-ates around an average position. Their functional properties depend on structural fluctuations and on the flexibility of certain parts of the molecule. The existence of internal movements within proteins is not a new concept. In 1955, LINDERSTRØM-LANG hypothesised that proteins have relatively flexible structures and fluctuate in solution; he described this mobility as the “breathing” of proteins. In order to con-firm this experimentally, he developed the methods of hydrogen/deuterium ex-change. A little later, as we described in Part IV, KOSHLAND proposed the induced-fit model to describe the conformational adjustments of an enzyme that occur upon binding of its substrate, allowing a better orientation of the atomic groups that react during catalysis. This image of a flexible enzyme has gradually superceded the fixed image of a lock and key as was first proposed. However, this dynamic vision of protein structures was neglected for over a decade and obscured by the static view of structures in three dimensions. Since the 1980s developments in nu-clear magnetic resonance spectroscopy have led to a reconsideration of the dy-namic aspects of protein structure in solution and have confirmed the hypothesis of LINDERSTRØM-LANG. The existence of movement within proteins has also been recognised by crystallographers. Crystal structures only represent time-averaged structures; the temperature factors analysed during the refinement of crystallog-raphic data have revealed larger structural fluctuations at the surface than in the protein core. Significant fluctuations have also been observed in the active sites of enzymes. More than twenty years of research has underlined the decisive role of these movements in enzymatic activity.
A great variety of internal movements occurs in proteins. These movements, of varying amplitudes, take place over a large time-scale, ranging from 10–14 s to several seconds. Vibrational movements correspond to times from 3 × 10–14 s to 3 × 10–12 s. Rotations about dihedral angles in the peptide backbone and protein side-chain have time constants around 10–8 s. Concerted movements with higher amplitudes occur over longer time periods. Proteins possess flexible and compact parts which ensure the transmission of movement across the molecule. For ex-ample, the relative rigidity of helices allows vectorial transmission of movement. Knowledge of the three-dimensional structure of an enzyme is a necessary but insufficient condition for understanding mechanisms of catalysis; an analysis of the internal dynamics of the molecule is also necessary. Local motion as well as
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CONCLUSIONS AND PERSPECTIVES
collective motion of high amplitude play an important role in enzyme catalysis. Their existence and analysis have been the subject of important experimental ap-proaches and theoretical developments over the last few years. Simulation methods using molecular dynamics, which involve applying NEWTON’s equations of motion to the different atoms in a molecule, have been adapted to the study of large mol-ecules and been made possible by the increased power and speed of computers.
If molecular enzymology had been for a while obscured by the spectacular progress in molecular genetics, it nowadays experiences a growing interest within the scien-tific community. On the one hand, knowledge of an increasing number of genomes stimulates research aiming to characterise the function of those proteins encoded by the genes; proteomics has superceded genomics. On the other hand, the devel-opment of theoretical studies during the 1990s linking quantum mechanics with molecular mechanics (QM/MM) has seen the study of the catalytic mechanisms of enzymes regain its former stature (FIELD et al., 1990). The strategy consists of applying quantum mechanical methods to those atoms reacting in the active site of an enzyme and molecular mechanical methods to the molecule as a whole. The application of these methodologies represents a very productive approach to under-standing catalytic mechanisms. Thus, several enzymes have been the subject of such studies, among which the aspartyl proteases including those from HIV, triose phosphate isomerase, lysozyme, methylene dehydrogenase, alcohol dehydrogenase, citrate synthase, thymidine phosphorylase, protein tyrosine phosphatase, choris-mate mutase, β-lactamase and uracil-DNA glycosylase. In most cases, these studies have helped to remove the ambiguity concerning the mechanism and structure of the transition state.
In parallel to the development of theoretical methods, emerging technologies are permitting the evolution of a reaction to be followed over ever shorter time-periods (of nanosecond and even picosecond order). Time-resolved crystallography, made possible by the power of synchrotron radiation, enables conformational kinetics to be monitored over times as short as a few hundred picoseconds. At the moment this method is limited to a few usable systems; its extension to the study of other mol-ecules rests with the possibility to trigger rapidly a synchronous reaction: all the molecules must be in the same state at the same time. Currently, we have at our disposal powerful tools which enable understanding of enzyme function in terms of the structure-dynamics-function relationship.
Besides the molecular aspects, developments in cellular enzymology in vivo are foreseeable in the coming years. The continual improvement in NMR spectroscopy in vivo offers an increasingly useful means to study metabolism and its regulation in cells, tissues or perfused organs and even in complete, living beings. The non-invasive character of the technique means that its use as a diagnostic tool is pro-gressing quickly. Although not yet widespread in fundamental research, in vivo NMR constitutes the most appropriate method for analysing metabolic control path-ways because it enables a precise measurement of the metabolite concentrations as
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MOLECULAR AND CELLULAR ENZYMOLOGY
a function of time. These approaches, linked to studies in vitro, offer huge potential for enzymatic studies which will be increasingly necessary with the deciphering of genomes, including the human genome. It is not enough simply to know the genes; it is vital to know the proteins for which they code and their functions, of which a large number remain as yet unknown.
An entire field of study is opening up with modern enzymology which will increas-ingly demand a multi-disciplinary approach. Knowledge progression will result from the synergy of groups of experts in specialities relating to physics, chemistry and most biological fields. Modern enzymology is therefore progressing along two complementary axes. The first is the high-resolution molecular aspect which in-cludes the temporal dimension of structure. The second is the cellular aspect which takes into account the biological context in which catalytic activity manifests itself and its many regulatory aspects in all their complexity.
BIBLIOGRAPHY
SPECIALISED ARTICLES
We provide here some references concerning the studies of motion within proteins in relation to their functional properties.
BRUCCOLERI R.E., KARPLUS M. & MAC CAMMON J.A. –1986– Biopolymers 25, 1767. BRUCCOLERI R.E. & KARPLUS M. –1985– Proc. Natl Acad. Sci. USA 82, 4995. CHOTHIA C., HUBBARD T., BRENNER S., BARNS H. & MURZIN A. –1997–
Annu. Rev. Biophys. Biomol. Struct. 26, 597. COLONNA-CESARI F., PERAHIA D., KARPLUS M., EKLUND H., BRÁNDÉN C.I. & TAPIA O.
–1986– J. Biol. Chem. 261, 15273. ECH-CHERIF EL KETTANI M.A., ZACKRZEWSKA K., DURUP J. & LAVERY R. –1993–
Proteins Struct. Funct. Genet. 16, 393. FIELD M.J., BASH P.A. & KARPLUS M. –1990– J. Comput. Chem. 11, 700. GUILBERT C., PERAHIA D. & MOUAWAD L. –1995– Comput. Phys. Commun. 91, 263. LINDERTRØM-LANG K.U. –1955– Chem. Soc. Spec. Pub. 2, 1. LINDERTRØM-LANG K.U. & SCHELLMAN J.A. –1959– Protein structure
and enzyme activity, in The Enzymes, 2nd ed., Vol. I, P.D. BOYER ed., Acad. Press, New York, 443–510.
MARQUES O. & SANEJOUAND Y.H. –1995– Proteins Struct. Funct. Genet. 23, 557. MOUAWAD L. & PERAHIA D. –1996– J. Mol. Biol. 258, 393. THOMAS A., FIELD M.J., MOUAWAD L. & PERAHIA D. –1996– J. Mol. Biol. 257, 1070. THOMAS A., FIELD M.J. & PERAHIA D. –1996– J. Mol. Biol. 261, 490.
766
GENERAL BIBLIOGRAPHY
SPECIALISED ARTICLES
Part I DODE P. –1956– Bases fondamentales et applications de la thermodynamique chimique,
Sedes, Paris. GLANSDORFF G. & PRIGOGINE I. –1971– Structure, stabilité, fluctuations, Masson, Paris. STRYER L., BERG J.M. & TYMOCZKO J.L. –2002– Biochemistry, 5th ed., Freeman Pub.,
San Francisco.
Part II BERTRANDIAS F. & BERTRANDIAS J.P. –1997– Mathématiques pour les sciences de la vie,
de la nature et de la santé, Collection Grenoble Sciences, EDP Sciences, Paris. PROTASSOV K. –2002– Analyse statistique des données expérimentales,
Collection Grenoble Sciences, EDP Sciences, Paris. RICARD J. –1973– Cinétique et mécanismes d’action des enzymes. I- Cinétique enzymatique,
Doin, Paris. SEGEL I.W. –1975– Enzyme kinetics, John Wiley & Sons, New York.
Part III CANTOR C.R. & SCHIMMEL P.R. –1980– Biophysical chemistry, Freeman Pub., San Francisco. GHELIS C. & YON J.M. –1982– Protein folding, Acad. Press, New York. JANIN J. & DELEPIERRE M. –1994– Biologie structurale, Hermann, Paris. LUNDBLAD R.L. & NOYES C.M. –1985– Chemical reagents for protein modifications,
Vol. I and II, C.R.C. Press, Boca Raton. MC PHERSON M.J. –1990– Directed mutagenesis: a practical approach,
Oxford Univ. Press.
Part IV CHRISTEN P. & METYZLER D.E. –1985– Transaminases, John Wiley & Sons, New York. FERSHT A.R. –1985– Enzyme, structure and function, 2nd ed., Freeman Pub., San Francisco. JENCKS W.P. –1969– Catalysis in chemistry and enzymology, McGraw Hill, New York. PELMONT J. –1996– Enzymes: catalyseurs du vivant, Collection Grenoble Sciences,
EDP Sciences, Paris. WALSH C. –1979– Enzymatic reaction mechanisms, Freeman Pub., San Francisco.
PHYSICAL CONSTANTS
Force: 1 dyne = 10–5 Newton = g . cm . s–2
Energy: 1 erg = 10–7 J = g . cm2 . s–2 1 J = 4.18 kcal
AVOGADRO’ number: N = 6.023 × 1023 mol
BOLTZMANN’ constant: kB = 1.381 × 10–23 J . K–1 = 3.298 × 10–24 cal . K–1
PLANCK’ constant: h = 6.626 × 10–34 J . s = 1.58 . 10–34 cal . s
FARADAY’ constant: F = 9.649 × 104 C . mol–1 = 2.306 × 104 cal . vol–1 . eq.–1
ABBREVIATIONS
C = coulomb
cal = calorie
K = Kelvin
eq. = equivalent
J = joule
s = second
g = gram
mol = mole
INDEX
A Abzyme, 439, 440, 536 Acetoacetate decarboxylase, 289 Acetoacetyl-ACP, 708 Acetyl-ACP, 708 Acetylation, 260 Acetylcholinesterase, 288, 293, 344, 362,
366, 412, 413, 434, 439, 447 Acetyl-CoA, 239, 666, 667, 700, 702, 706 Acetyl glucosamine, 648 Acid aspartic, 231, 312 glutamic, 231, 312, 646, 656 ketonic, 519 lipoic, 701, 705 muramic (NAM), 497 Actin, 736 Activation, 97, 137, 139–141, 254, 441, 453,
454, 472, 480, 501, 547, 573, 578, 602, 617, 621, 629, 676
allosteric, 556 of complement, 253, 254 energy, 97, 367, 394, 426, 427, 443 partial, 139 by the substrate, 141, 446 total, 137 volume, 222 Activator, 122, 136, 137, 147, 468, 496, 549,
550, 552, 558, 560, 579, 584, 672, 707
Active site (centre) of enzymes, 11, 103, 264, 267, 270, 280, 331, 373, 416, 457, 460, 461, 469, 471, 472, 481, 482, 484, 496, 498, 506, 511, 524, 530, 531, 533, 536
Activity, 21, 233, 234, 245, 267–271, 323, 324, 327, 328, 561, 612, 641, 653, 717
coefficient, 21 molar, 107 Acylation, 150, 151, 273, 278, 302, 308, 310,
314, 415, 447, 462
Acyl carrier protein (ACP), 1, 707–709 Acyl-enzyme, 149, 152, 278, 279, 361, 420,
447, 448, 458, 459, 463–465 Adenine, 233, 246, 435, 505 Adenosine deaminase, 293, 434, 439 Adenylate, 246, 435–438 cyclase, 261, 620, 621 kinase, 760 transferase, 652, 653 Adenylation, 649, 652, 653 ADP, 54, 260, 580, 582, 622, 644, 662, 666,
734, 736 polyribosylation, 260 ribosylation, 260, 261, 641, 644, 647 ADP-Mg, 182 ADP-ribose, 260, 645 Adrenaline, 621 Affinity, 20, 57, 64, 68–70, 112, 137, 139,
141, 149, 553, 554, 556, 557, 559, 562, 569, 584, 587, 592
chemical, 68–70 Alanine, 231, 341, 368, 479, 489, 671, 703 Alcohol dehydrogenase, 166, 168, 501–507,
715 Aldehyde, 507 Aldimine, 521, 522, 524, 525 Aldolase, 526–534, 712, 713, 736 class I, 527 class II, 527 KDPG, 527, 528, 531 Aldose-ketose isomerases, 409, 410 Alkylation, 302, 307, 308, 310, 314 Allosteric effector, 549, 559, 572, 597, 599, 646, 652,
653, 662, 666, 667, 736, 754 model concerted, of MONOD, WYMAN,
CHANGEUX, 553–560, 578 generalised, 567 sequential, of KOSHLAND, NEMETHY,
FILMER, 561–566 Allostery, 548, 572, 698, 707 Amido transferase, 695
Amino acids, 232, 242, 244, 281, 284, 302, 331, 332, 518–520, 526, 531
hydrophobic, 295, 528 Amino-acyl adenylate, 489 AMP, 233, 298, 402, 403, 573, 574, 578, 579,
582, 584, 585, 619, 644 cyclic (cAMP), 77, 259, 298, 620–623,
647, 667, 760 Amylase, 234, 260 Analysis, 323 statistical data, 61, 120, 183 Anthranilate, 680, 685, 686 synthase, 334 Anti-cooperativity, 548, 563, 564, 606–609,
745–747 Apoptosis, 673, 674, 676 Apparatus continuous-flow, 202, 203 GOLGI, 727, 729 quenched-flow, 205 stopped-flow, 204, 205, 224, 225 Approximation of quasi-equilibrium, 112, 132, 137, 175 of the steady state, 174 Archaebacteria (Archae), 241, 671 Arginine, 151, 260, 320, 642, 646, 737 Arylation, 302, 307, 308, 310, 314 Asparagine, 260, 480, 583, 647 Aspartate, 194, 313, 362, 463, 585–587,
591, 593 amino transferase, 179–181, 270, 272, 289,
518–526 2-oxoglutarate amino transferase, 179,
289 transcarbamylase (ATCase), 194, 220, 362,
365, 433, 585–595, 695, 737 Aspartokinase, 602 Aspirin (hydrolysis of), 414, 424 Association, 733–736 enzyme-substrate, 267, 268, 272, 348, 360,
365, 369, 370, 374, 474, 485 ATP, 29, 201, 202, 287, 366, 402, 434–437,
573, 579, 580, 582, 584, 585, 592–595, 599, 600, 619–621, 641, 662, 667, 672, 697, 734–737
hydrolysis of, 29–32 ATPase, 298, 625, 673 ATP-Mg, 182, 183, 490, 491–493, 496, 611,
612, 622, 697 B Binding, 1, 2, 3 complement, 659
ligand, 51, 282 polynome, 50 Biosynthesis, 649, 650, 695, 738, 754 Biotin, 411, 699, 707 Blood clotting, 253, 259, 629, 637, 654, 655,
659 Bond covalent, 284, 360 π, 360 glycosidic, 232, 258, 279, 644 hydrogen, 282, 358, 359, 577, 643, 644,
676, 684, 698, 711 peptide, 232, 242, 282, 632, 656, 658 Butyryl-ACP, 708 C C6-acyl and C16-acyl-ACP, 709 Calmodulin, 623–625 Carbamyl aspartate, 194 phosphate, 194, 585, 591, 697–699, 737 synthetase (CPSase), 695, 697, 700, 737 Carbamylation, 194, 293, 303, 585, 586 Carbanion, 379, 388, 405, 407, 408, 410, 512,
532, 701 Carbene, 295, 296, 298 Carbocation, 379, 405, 431, 499 Carbodiimide, 312, 313 Carbonic anhydrase, 412, 413 Carboxylation of glutamyl residues, 259 γ-carboxyglutamyl, 259 Carboxypeptidase, 361, 362, 366, 453,
478–489, 641 CARNOT’s principle, 63 Caspase, 673–677 Catalase, 344 Catalysis, 101, 280, 335, 384 chemical, 377, 378 electrophilic, 387 enzymatic, 377, 432 general acid, 389, 390, 392, 477, 499, 512,
676 general acid-base, 391, 392, 478, 512,
517 general base, 385, 392, 445, 486 heterogenous, 101 homogenous, 101 intermolecular, 413–416 intramolecular, 413–415 monofunctional, 424 nucleophilic, 380, 384 polyfunctional, 389, 423, 424 Catalyst, 4, 100, 101, 377, 387, 390, 515, 516
MOLECULAR AND CELLULAR ENZYMOLOGY II
Catalytic efficiency, 343, 362, 414, 427, 433, 448 triad, 363, 457, 462, 470, 616 Cell, 725, 727–732, 735 Cellular enzymology, 725 Cell wall, 733, 736 plant, 736, 747 growth of, 748 Channelling, 679, 681, 687, 691, 694, 697,
699, 705, 712, 717, 718 Chemical kinetics, 85 first order, 88, 89 pseudo-first order, 92 second order, 93, 94 zero order, 96 Chemical labelling, 280 affinity, 290–295 by a coenzyme, 286–290 differential, 301 direct of amino-acid carboxylates (aspartate
and glutamate), 312–313, 314 of α- and ε-amino groups, 302–308 of arginine, 320 of carboxyl groups, 312–313 of histidine, 308, 309 of hydroxyl groups (serine and
threonine), 314–318 of methionine, 319 of sulfhydril groups, 314–318 of tryptophan, 321 of tyrosine, 310, 311 photoaffinity, 295–298 principle of, 280 by a quasi-substrate, 286–290 by suicide reactants, 299 Chemical modifications (post-translational),
257–261 Chirality of biological molecules, 247 Chloramphenicol acetyl transferase, 704 Chorismate, 680 Chromatography, 55 Chymotrypsin, 153, 255, 256, 286, 288, 291,
292, 299, 345, 366, 388, 420, 428, 429, 447, 452–465
Chymotrypsinogen, 255, 456–457 Citrate, 707 synthase, 718 CLELAND nomenclature of, 167, 184 rules of, 177 Coefficient control, 750 elasticity, 750, 751
electrostatic interaction, 43 phenomenological, 71, 74 of transmission, 99 Coenzyme, 166, 387, 388, 406, 407, 519,
522, 700, 715 A (CoA), 239, 241, 705 Colipase, 613, 614, 618 Compartmentalisation, 730, 738 cellular, 727, 730 metabolic, 737, 738 Complex, 335, 633, 635, 637, 638, 659, 661,
670–673, 730 enzyme-activator, 137 enzyme-inhibitor, 132, 498 enzyme-substrate, 105, 127, 132, 137, 155,
171, 206, 335, 347, 361, 365, 369, 420, 441
ES, 137, 154, 422, 424, 431 intermediate, 6, 347, 431 lipase-colipase, 613 of lysozyme with diverse inhibitors, 498 MICHAELIS, 150, 274, 276, 278, 279, 348,
360, 361, 365, 407, 413, 422, 424, 426, 444, 448, 458, 459, 461, 525
multi-enzyme, 679, 700, 702, 707, 717, 737, 749
tetrahedral, 381, 461, 477 Concentration, 731, 752 reduced local-substrate, 743, 744 reduced substrate, 548, 555, 743 Conservation of energy, 65 of mass, 48, 54, 64, 68 Constant, 556 affinity, 553, 562, 633 allosteric, 222, 553, 557, 601 BOLTZMANN’s, 43, 75, 99 catalytic (kcat), 112, 139, 141, 155, 159,
430, 435, 474, 489, 554, 556, 559, 570, 583, 633, 662, 686
dielectric, 21, 43, 273, 280, 349, 441, 444 dissociation, 111, 112, 130, 132, 137, 141,
154, 158, 169, 171, 196, 367, 382, 389, 391, 395, 434, 438, 554, 555, 559, 570, 618, 624, 744
equilibrium, 21, 23, 26, 27, 73, 209, 221, 400, 408, 433, 606, 715
inhibition, 130, 361, 366, 460 intrinsic association or microscopic, 38, 40,
44, 50 ionisation, 274, 277, 278 kinetic, 151, 181 MICHAELIS (Km), 106, 112, 123, 127, 130,
134, 138, 149, 154, 155, 166, 169,
INDEX III
172, 196, 198, 269, 275–279, 361, 400, 459, 488, 717
PLANCK’s, 99, 356 specific rate, 7, 164, 207, 211, 212, 222,
279, 290, 301, 323, 327, 382, 400, 433, 446, 512, 694
Constraints, 373 diffusional, 745 electrostatic, 745 Control, 749 of linear metabolic pathways, 749–755 of metabolic cycle, 755–761 Cooperativity, 44, 46, 248, 548, 550, 552,
559, 571, 573, 585, 600, 606, 607, 608–611, 745–747
Coupling, 248 diffusion-reaction, 741, 743–745 energetic, 27, 30 fundamental equation, 741 thermodynamic, 70, 567, 569 Crotonyl-ACP, 708 Cryoenzymology, 220 Cyanogen bromide, 319 Cycle cellular, 676, 739 futile, 755 KREBS, 82, 518, 727, 731, 737, 754 limit, 758 metabolic, 755 pentose, 754 tricarboxylic, 22 urea, 518 Cysteine, 244, 281, 314–318, 380, 468, 470,
471, 586, 642, 691, 694, 710 Cystine, 318 Cytidine deaminase, 412, 432, 433 Cytochrome, 508, 509 c, 512, 513, 675 Cytoplasm, 646, 731–733, 739 Cytoskeleton, 624, 670, 676, 731, 735, 736 Cytosol, 670, 706, 712, 729 D DATP, 599, 600 DCTP, 599, 739 Deacylation, 151, 152, 154, 156–158, 278, 415,
416, 420, 444, 447, 458, 463, 465 Deamination, 308 Decarboxylase, 412 Dehydrogenase, 81, 364, 372, 378, 406, 491,
501 alcohol, 501–507 glucose-6-phosphate, 148, 183, 201, 330
α-ketoglutarate, 706 lactate, 366, 445, 502, 534, 535 malate, 502 pyruvate, 666, 700, 702, 704 Denaturation, 24, 269–271 irreversible, 269, 270 reversible, 270 Desoxyribonuclease, 632 Desoxyribonucleotides, 596, 599, 600 Dialysis dynamic, 52, 53, 584 equilibrium, 51, 179 Diffraction neutrons, 463, 534 X-rays, 10, 268, 463, 478, 534 Diffusion, 115, 399, 743–745 rate, 514 substrate, 115, 741 Dihydrofolate reductase, 718 Dihydrolipoamide acetyl transferase, 700 dehydrogenase, 700 Dihydroorotase (DHO), 695 Dihydroxyacetone phosphate, 513, 514, 516, 518 Diisopropyl phosphofluoridate (DFP), 288 Dimerisation, 216 1,3-diphosphoglycerate, 490, 496 Dipole, 341, 348, 350, 352, 357, 378, 441 induced, 353, 354, 363, 364 oscillating, 355 permanent, 351, 355 Disulphide bridge, 257, 464, 467, 484, 614,
635, 636, 660 DNA, 1, 244, 245, 249, 251, 331–333, 727,
729–733, 739 ligase, 729 polymerase, 332, 729 Domain (structural), 344, 491, 493–495, 502,
507, 509, 510, 586, 590, 681, 685, 691, 698, 699, 711, 734
catalytic, 502, 504, 658, 703, 704 coenzyme binding, 502, 503, 508 DONNAN effect, 51 equilibrium, 52 DTTP, 739 Dynamics, 734, 764 molecular, 446, 447, 448, 478, 495 structural, 764 E EADIE plot, 60, 116, 117, 123, 124, 126–129,
133–135, 143
MOLECULAR AND CELLULAR ENZYMOLOGY IV
Effects cooperative, 45, 46, 547, 550, 585, 587,
591 diffusional, 730 electrostatic, 283, 284, 441, 443, 745 entropic, 421 heterotropic, 554, 556, 585, 592, 593 homotropic, 554, 556, 592 isotope, 393–401, 478, 507, 514, 515, 517,
534 magnitude of, 399 primary, 393, 394 secondary, 396, 397, 398 with tritium, 395 orientation, 416, 418, 419 solvent, 395, 396, 397 steric, 283, 284, 398 Elastase, 150, 428, 452, 453, 458, 629, 632,
634, 637, 638, 658 Electron, 510, 512, 538 microscopy, 618, 710 Electrophile, 377, 379, 380, 387, 477, 507,
519 Endergonic, 20, 27, 33, 80 Endopeptidases, 465 Energetics, 1 of enzyme-substrate associations, 365 Energy, 349, 358, 359, 366–368, 384, 385,
395, 425–428, 447, 501, 518 activation, 97, 101, 367, 426–428, 570,
573, 606, 610 association, 426, 489 barrier, 98, 99, 377, 380, 425 binding, 499 change, 20, 24, 25 free, 19, 20, 23, 46, 49, 69, 365, 552, 568,
569 interaction, 349, 354, 369, 551, 569 enzyme-substrate, 46, 49 internal system, 16 potential, 350–353, 355–357 standard free, 20, 21 Enolase, 713 Enoyl-ACP hydratase, 708 reductase, 708 Enthalpy, 277, 365, 366, 369 change, 16, 20, 22–24, 66, 100, 209, 217 Entropy, 15–20, 33, 66–70, 99, 237, 366, 370,
413, 419–422 Enzyme allosteric, 547, 549, 572, 749, 753, 759 angiotensin-converting, 478 condensation, 708, 710
immobilised, 741, 745, 748 kinetics of reactions catalysed by, 743 interconversion, 653, 654, 662, 663, 667,
670 membrane, 730, 733 mnemonic, 602, 605–611 oligomeric, 532, 555, 561 plant cell wall, 736, 747, 748 Equation ADAIR, 48, 552, 567 HILL, 47, 550, 551 LINDERSTRØM-LANG, 44 MICHAELIS, 106, 111, 115, 150, 184, 186,
188 integrated, 118, 143, 196 Equilibrium, 105, 207, 360, 553–554, 562,
568, 611, 759 conformational, 209, 271, 554, 557, 563 far from, 63, 64, 74 protein-ligand association, 37 thermodynamic, 101, 570 Esterases, 153, 439, 447 Eubacteria, 241 Eukaryotes, 241, 670, 671, 672, 729 Evolution, 2, 229, 230, 241, 242, 262, 333,
452, 601, 671, 673, 697, 712, 761 protein, 2 species, 2 time-scale, 229, 230 Exchange hydrogen/deuterium, 764 isotopic, 515 Exergonic, 20, 29, 365, 369, 374 Exon, 235, 245, 503 Exopeptidases, 670 F Factor blood clotting, 253, 259 probability, 38 signal amplification, 664 statistical, 46 tissue, 655, 656 VON WILLEBRAND, 655 FAD-FADH2, 27, 81 Fatty acid synthase, 1 Fibrin, 253, 656, 658 Fibrinogen, 253, 656, 659 FICK’s first law, 742 second law, 742 Flavin, 405, 406, 501, 507–510, 512 Flavocytochrome b2, 407, 501, 507–513
INDEX V
Flow methods continuous, 202 quenched-flow, 205 stopped-flow, 204 Fluctuations, 79 local, 70 Flux, 71, 72, 666, 730, 750, 751, 753, 755 ATP, 666 matter, 72 metabolic, 73, 82, 679, 715, 719, 749 substrate, 73 FMN, 81, 507, 709 Focus stable, 78, 758 unstable, 78, 758 Force, 348 electrostatic interaction, 348–353, 357 induction, 353, 357 LONDON’s dispersion, 348, 355 Fructose-1, 6-diphosphate, 526, 527, 579 Fructose-6-phosphate, 734 Fructose bisphosphate, 736 Fumarase, 141 Function catalytic, 3 linked, 48 motility, 3 photoreceptor, 3 regulatory, 550, 554 saturation, 41, 50, 106, 107, 555, 563 of state, 554 transport, 3 G α-galactosidase, 345 β-galactosidase, 140, 141, 153, 161, 162, 165,
195, 206, 221, 295, 345, 366, 429, 432
Gene, 503 fusion, 502, 685, 696 GIBBS (free energy), 262 Glucagons, 621 Glucanase, 748 Glucokinase, 201 Glucose, 30, 80, 182, 404, 573, 611, 612,
734, 737, 754 Glucose-1-phosphate, 575, 648, 754 Glucose-6-phosphate, 181, 183, 201, 287,
330, 404, 573, 578, 611, 644, 754, 755
Glutamate, 361, 363, 378, 680, 691, 738 Glutamine, 334, 680, 695, 697, 699, 705 synthetase, 649, 653, 669
Glutarate, 180, 181 Glutaredoxin, 596 Glutathione, 596 Glyceraldehyde-3-phosphate, 334, 364, 490,
513, 515, 516 dehydrogenase, 297, 364, 380, 502, 566,
572, 712–715, 733, 734, 736 Glycerolphosphate dehydrogenase, 713 Glycine, 231, 361, 394, 460, 479, 673 Glycogen, 573, 642, 730, 754, 755 phosphorylase, 220, 234, 259, 417,
573–578, 602, 642–644, 669 See also Phosphorylase a and b synthetase, 642 Glycolate oxidase, 509, 510 Glycolysis, 727, 730, 759, 761 Glycoproteins, 465, 632, 633, 656, 657 Glycosidases, 431, 432, 447, 496, 501 Glycosyl enzyme, 499, 501 groups, 404 Glycosylation (N- and O-), 258, 647–649 β-glycosyl transferase, 404, 602, 648 Graphical plot ARRHENIUS, 100 DIXON, 124, 126, 133–135 EADIE, 60, 124, 566 EISENTHAL and CORNISH-BOWDEN, 118, 119 HANES-DIXON, 118 HILL, 48, 551 from integrated rate equation, 118, 131 KLOTZ, 40, 60 LINEWEAVER-BURK, 60, 124, 566, 606, 609,
612 SCATCHARD, 40–42, 46, 60 semi-logarithmic, 116 VAN T’HOFF, 100 Graphs (method of), 145–148 GTP, 245, 673 H Haem, 238, 508, 509, 512, 559 Haemoglobin, 10, 203, 220, 264, 550, 552,
553, 734, 763 HALDANE, 109, 170 relation, 108 Heat (CLAUSIUS’ non-compensated), 69 α helix, 446, 452, 467, 480, 491, 513, 528,
577, 597, 598, 622, 624, 625 Heparin, 657, 658 Hexose, 247 High pressure, 223, 224 enzyme reactions under, 221
MOLECULAR AND CELLULAR ENZYMOLOGY VI
HILL, 551 coefficient (number), 47 diagram, 47 equation, 47, 550 number, 47, 606 Histidine, 222, 244, 280–282, 297, 307–309,
363, 378, 380, 456, 458, 463, 470, 471, 479, 484, 493, 509, 515–517, 522, 642, 704
Homeomorphic reaction schemes, 177 Homeostasis, 725 Hormone, 253, 620, 632, 637, 647 adrenocorticotropic, 254 β-endorphin, 254 enkephalin, 254 lipotropic (β-LPH), 254 melanocyte-stimulating, 254 Hydrolysis, 132, 141, 142, 152, 161, 165,
166, 233, 245, 273, 303, 316, 319, 363, 389, 401, 414, 420, 424, 428–431, 496, 666
acid, 315 enzyme, 166 Hydrophobic, 272, 445, 476, 479, 485, 492,
509, 529, 530, 532, 594, 616, 621, 635, 639, 660, 684, 699, 712, 733, 734
interaction, 263, 359, 363 Hydroxylation of lysines, 258 of prolines, 258 Hysteresis loop, 743 I Imidazole, 222, 224, 233, 280, 308, 309, 378,
380, 415, 416 IMP, 573 Inactivation, 285, 322, 650 irreversible, 670, 691 Indole, 334, 687, 690, 695, 697 Indole-3-glycerolphosphate (IGP), 680 synthase, 685, 686 Indole propanolphosphate, 691 Induced fit, 370–373, 422, 423, 561 Induction force, 357 Inhibition, 122, 124, 125–136, 273, 299, 300,
361, 480, 584, 602, 641, 734, 744, 747
allosteric, 556, 557 competitive, 122, 124, 549, 736, 745 non-competitive, 125, 126 partial, 132 competitive, 133
mixed, 135 non-competitive, 134 by the reaction products, 130, 131 by the substrate, 129 total, 122 uncompetitive, 127, 128 Inhibitors, 122, 124–127, 147, 172, 177, 178,
218, 256, 273, 288, 290, 322, 361, 362, 366, 371, 431, 432, 523, 549, 550, 556, 557, 559, 584, 632, 641, 661, 672, 684, 691, 698, 744, 745, 752
allosteric, 560, 589, 734 dead-end, 177, 178 protease, 632, 637, 641 serine protease, 288, 632 bovine pancreatic (BPTI), 461, 633, 634 hirudine, 638, 639 irreversible, 122 serpins, 633, 637, 638, 658, 673 antiplasmin, 637 α1-antiprotease, 632, 637 ovalbumin, 637 plakalbumin, 637 soybean, 634 of thiol proteases, 639, 640 bromelain, 640 cathepsin B1, 640 cystatin, 640 stefin, 640 Insulin, 10, 254 Interactions, 55, 348–357, 492, 511, 562, 563,
589, 612, 623, 625, 731, 733 electrokinetic, 348, 355, 357 electrostatic, 42, 43, 272, 284, 348, 357,
358, 495, 518, 526, 734, 746 of enzymes with cell components, 733 hydrophobic, 1, 644 induction, 353 KEESOM, 353 repulsive, 356 VAN DER WAALS, 348, 358, 362, 368, 461, 643 Intermediate, 381 reaction, 206 tetrahedral, 381, 401, 402, 458, 461, 463,
477, 488, 635, 676 Intron, 245, 503 Iodation of histidine, 308 of tyrosine, 260, 308, 310 Iodoacetamide, 314, 319 Ion, 348, 350, 353, 377, 379 hydride, 507 oxonium (oxyanion), 461
INDEX VII
Ionic strength, 21, 43, 52, 273, 362, 455, 495, 719, 734, 746, 747
Ionisation, 270, 274, 276, 278, 279 Isoenzymes, 521, 527 Isoleucine, 270, 295, 361, 368, 738 Isomerisation, 214, 217 K Kcat/Km (ratio), 277, 279, 367, 412, 413, 422,
423, 426, 428–430, 434, 436, 440, 633, 687
Ketone, 307 Kinase, 259, 298, 372, 490–496, 666, 729 adenylate, 31, 760 creatine, 166, 490 hexokinase, 181–183, 199, 200, 268, 404,
417, 490 phosphofructokinase, 259, 490, 572,
579–585, 734, 736, 759 phosphoglycerate, 490, 713, 715 protein kinase, 490, 620–623, 676 See also Phosphorylation protein kinase C (PKC), 733 pyruvate, 200, 330, 490, 713, 759 Kinetics, 582, 605, 608, 609 enzyme, 484 KING and ALTMAN, 168, 172, 174 graphical method of, 143, 144 KLOTZ plot, 40 Km, 717 L β-lactamase, 299, 300, 334, 413, 765 Lactate, 502, 534, 535 Leucine, 361 LINEWEAVER-BURK plot, 60, 117, 120, 123, 124,
126–129, 133–135, 566, 606, 609 Lipase, 613–619 Lipoamide, 702, 703, 705 Localisation (cellular), 642 Lysine, 151, 258, 272, 289, 290, 305, 307, 330,
460, 472, 484, 520–522, 526, 531, 642, 646, 673, 684, 688, 701, 705, 712
Lysosomes, 727, 729 Lysozyme, 10, 372, 373, 425, 431, 481,
496–500, 763, 765 M Malate, 535 Malonyl-CoA, 707 ACP transacylase, 711
Mannose, 648, 661 Mass action (law of), 7, 27, 86, 89, 91, 93 spectrometry, 501 Mechanism, 375, 436, 438, 462, 507, 511,
512, 514 Bi Bi, 166 iso-ordered, 167 ordered, 167, 168, 170, 171 ping-pong, 168, 172, 173 random, 168, 174–176 catalytic, 364, 377, 459, 471, 477, 479,
486, 487, 495, 496, 499, 506, 524, 532, 671
ping-pong, 168, 172, 173 random Bi Uni, 138 Ter Bi, 166 Ter Quad, 166 THEORELL-CHANCE, 168 Uni Bi, 166 Membrane, 34, 235, 236, 646, 647, 655,
659–661, 710, 728, 730, 731, 733, 734, 736, 737, 741, 746
plasma, 727 Metabolism, 343, 412, 662 cellular, 240, 519, 545, 649, 727, 733 theory of, 749, 755 regulation of, 136 semi-cellular, 241 at a surface, 236 Metabolites, 727, 751 compartmentalisation of, 737, 738 flux of, 749, 750, 754 Methionine, 244, 252, 280, 282, 285, 290,
294, 314, 319, 479, 685 Methods chemical, 534 for studying enzyme reactions, 193 continuous, 194 discontinuous, 194 flow, 202–205 potentiometric, 195 relaxation, 206 chemical, 207 spectrum of, 211 thermal (T-jump), 209 MICHAELIS’ law, 233, 743 Microenvironment, 441 Microtubules, 736 Mitochondria, 712, 727, 729, 731 Molecular chaperones, 263 modelling, 286, 333, 369, 425, 434 Muscular contraction, 641
MOLECULAR AND CELLULAR ENZYMOLOGY VIII
N N-acetyl-glucosamine (NAG), 372, 431, 497,
498, 500 NADH-NAD, 238, 239, 363, 364, 378, 406,
490, 491, 501, 504–506, 666, 711–714, 734, 737, 759
NADPH-NADP+, 239, 406, 709 NAM, 239, 431, 497–499 N-ethyl maleimide (NEM), 315, 334 Nicotinamide, 712 Nitrene, 295, 298 Node stable, 758 unstable, 758 N-phosphonacetyl-L-aspartate (PALA), 365 Nuclear magnetic resonance (NMR), 363,
634, 638, 754, 755, 765 Nucleic acids (abiotic formation of), 233 Nucleophile, nucleophilic, 153, 154,
156–162, 280, 281, 284, 288, 302, 306, 307, 377, 379, 380–387, 401, 402, 414, 425, 458, 462, 671, 711
attack, 383, 410, 425, 477, 488, 496, 524 competition, 153, 157, 159–166 Nucleotides, 592, 593, 624, 737, 738 abiotic formation of, 233 purine, 592 pyrimidine, 592, 649 Nucleus (cell), 646, 670, 676, 727, 729,
737 Number AVOGADRO(’s), 354 HILL, 551 O Orbital, 418 Order, 85 global, 86 and molecularity, 97 partial, 86 of a reaction, 86, 87 with respect to concentration, 88 with respect to time, 87 Original soup hypothesis, 231 Ornithine, 698 transcarbamylase (OTCase), 737 Oscillations, 760, 761 amplified, 77, 758 damped, 758 glycolytic, 77, 759 in the membrane potential, 760 periodic, 77, 79
sustained, 760 temporal, 79 Oxaloacetate, 344, 520, 718 Oxidation, 30, 32, 258, 310, 501, 507 2-oxoglutarate, 526 Oxyanion, 461, 471, 477, 635, 676, 698 Oxydoreduction, 258, 404–406, 501 potential, 27, 28 reactions, 81 P Palmitate, 709 Palmitoylation, 261 Papain, 153, 425, 446, 465–470, 639 Parachloro mercuribenzoate (PCMB), 315 Parameters, 169, 440 kinetic, 107–109, 116, 131, 149, 150,
156–158, 166, 170, 268, 275, 278, 427, 429, 430, 437, 536, 685
thermodynamic, 374, 420 Paranitrophenyl acetate, 286, 384, 415, 416 guanido benzoate, 286 Pectin, 748 Pepsin, 366, 425, 428, 471–473, 476 Pepstatin, 475 Peroxidase, 221, 310, 344, 378, 379 pH, 195, 233, 235, 269–279, 361, 386, 389,
392, 425, 462, 472, 476, 484, 506, 591, 633, 686, 716, 736, 746, 748
effect of on the conformational state, 270, 271 on the ionisation state of catalytic
groups, 274 profile, 275, 392 Phase latent, 140 pre-steady state, 104 steady state, 104, 140 Phase plane, 758 Phenylalanine, 361, 469, 479 Phenyl methane sulfonyl fluoride (PMSF),
288, 289 Phosphatase, 259, 380, 641, 654, 666 acid, 380, 747 alkaline, 380, 412, 434, 447, 727 Phosphate, 490, 492, 496, 684, 685, 699, 754 pyridoxal, 179, 180, 238, 289, 290, 331,
387, 388, 519, 520, 523, 577, 642, 687, 692
pyridoxamine, 179, 180, 519–522 Phosphatidylinositol, 734 Phosphatidylserine, 734
INDEX IX
Phosphocreatine, 754 Phosphodiesterase, 245, 646 Phosphoenolpyruvate, 408 Phosphofructokinase, 259, 490, 572,
579–585, 734, 736, 759 Phosphoglucomutase, 287, 713 3-phosphoglyceraldehyde, 32 3-phosphoglycerate, 32, 408, 490, 493–495,
526, 527 Phosphoglycerate kinase, 268, 490–496, 713 Phospholipase, 363, 632, 734 Phospholipids, 655, 656, 733, 734 Phosphopantethein, 707 Phosphoribosylanthranilate, 680, 685 isomerase-indoleglycerol synthetase (PRA
isomerase-IGP synthase), 679, 681, 686
isomerase (PRA isomerase), 680, 685 Phosphoribosyl pyrophosphate, 238, 239 amido-transferase, 699 Phosphorylase a and b, 287, 573–579, 668, 730 Phosphorylated components, 30, 31 thermodynamic scale of, 31 Phosphorylation, 259, 287, 363, 573, 577,
629, 641, 662, 668, 754, 755 oxidative, 82, 727, 737 Phosphoryl-enzyme, 287, 447, 490, 496 Phosphotribose, 237, 242 Photooxidation, 309 Photosynthesis, 80, 81 pK, 146, 222, 271–273, 276–279, 383, 389,
445, 463, 476, 481, 526, 591 apparent, 49 Plot primary, 170, 171, 173, 176 secondary, 171, 173 Polarisability, 354, 356
α0, 354 Potential, chemical, 22, 68 Prebiotic chemistry, 231 Pressure, 210, 221, 223, 224, 691 Procolipase, 614, 615, 617 Prohormone, 252 pro-ACTH, 254 proinsulin, 254 Prokaryotes, 253 Proline, 432, 682, 703 racemase, 408, 432 Protease, 108, 288, 344, 380, 446, 452–489,
633, 634, 658, 676, 696 aspartyl, 471–478 chymosin (rennet), 471 endothiapepsin, 473
gastricin, 471 penicillinopepsin, 471, 473, 475, 476 pepsin, 471, 473 rennin, 471, 472 rhizopuspepsin, 473 cysteine, 153 metalloproteases, 378, 478–489, 641 serine, 149, 151, 153, 166, 221, 254, 271,
286, 288, 331, 380, 415, 425, 427, 452–465, 632, 634, 655
chymotrypsin, 150, 153, 166, 255, 256, 271, 286, 292, 331, 344, 345, 366, 415, 417, 420, 428, 429, 446, 447, 451, 453–465, 629
elastase, 428, 452, 463, 629, 632, 634, 637, 658
α-lytic, 452 subtilisin, 452, 629, 634 trypsin, 150, 151, 163, 166, 218, 219,
271, 286, 292, 331, 345, 363, 429–431, 452, 455, 629, 634, 636, 658, 660
thiol, 166, 446, 465–471, 632, 673 actinidine, 465, 466, 468, 469 bromelain, 465, 466, 468, 469, 640 cathepsin, 465, 466, 468, 469, 640 chymopapain, 465, 466, 468 papain, 446, 465–470, 639 Proteasome, 670 20s, 671, 672 26s, 672 Protein CAD, 679, 695–697 Proteolysis, 128 limited, 251, 254, 472, 613, 629, 633, 654,
656 Prothrombin, 259, 655, 657 Protobiont, 234 Purine, 231, 232, 242 Pyridoxamine, 519, 522 Pyrimidine, 231, 232, 242, 243, 490, 509,
649, 695 Pyrophosphate, 402 thiamine, 238, 388, 404, 444, 700 Pyruvate, 666, 737 decarboxylase, 232, 233, 700 dehydrogenase, 666, 679, 700, 701,
702 kinase, 490, 528, 713, 759 Q Quasi-equilibrium, 7 approximation, 112, 132, 139, 175, 176
MOLECULAR AND CELLULAR ENZYMOLOGY X
R Racemase, 408, 432 Rate, 730, 749, 751 average, 85 diffusion, 412, 413, 514, 731, 743 initial, 8, 113, 114, 130, 154 maximal, of reaction (Vm), 106, 107, 123,
656 reaction, 115, 116, 556, 611, 743, 752,
753 Reaction, 297, 303, 307, 314–316 bimolecular, 72, 213, 214, 218 coupled, 27 elimination, 408 enzyme, 400, 409, 425 one substrate, 137, 143, 148 two substrate, 132, 166 first order, 88, 89 irreversible, 287 isomerisation, 214 pseudo-first order, 92 rate of, 556, 611 rearrangement, 408 reversible, 90 second-order, 93, 94 transfer, 451 acyl, 402 of glycosyl groups, 404, 405, 451 of phosphoryl groups, 402, 451, 489 zero-order, 96 Reactivity, 160, 382, 441 Regression multilinear, 186 non-linear, 120, 184, 186, 188 simple, 184 Regulation, 251, 259, 261, 547, 618, 623,
629, 641, 642, 644, 647, 655, 657, 661, 667, 670, 677, 715, 725, 736, 749
allosteric, 547, 573, 579, 581, 599, 642, 654
of cell metabolism, 733 covalent, 629, 677 of enzyme activity, 8, 259, 679 metabolic, 136, 573, 730 non-covalent, 626 by phosphorylation, 641, 642 Relaxation thermal, 209–219, 498 times, 213, 217 Replication, 729 Reticulum (endoplasmic), 252, 253, 727, 729
Retroinhibition, 248 Reversibility, 108 microscopic, 177 Ribonuclease, 153, 262 Ribonucleotide reductase, 596–601, 729 Ribonucleotides, 596, 600 Ribose, 492, 505 Ribosomes, 236, 252, 727–730 RNA, 245, 246, 249 mRNA, rRNA, sRNA and tRNA, 248, 368,
369, 434–436, 438, 489 polymerase, 259 S Saddle point, 77, 78 Salt bridge, 256, 271, 455, 456 SCHIFF base, 289, 290, 360, 388, 392, 688 Serine, 254, 256, 280, 286–288, 290, 293,
314, 331, 368, 378, 380, 423, 452, 458, 462, 464, 634, 639, 642, 643, 647, 648, 687, 690, 707
Serpin, 633, 637, 638, 673 β sheet, 597, 613, 614, 622, 637, 638 Signal peptidase, 252 peptide, 252, 727 Site (centre) active, 57, 58, 256, 264, 285, 286, 288,
290, 295, 314, 322, 331, 334, 335, 337, 363, 373, 413, 444, 445, 447, 457, 460, 461, 464, 471, 472, 481, 484, 485, 488, 496, 498, 506, 511, 524, 530, 531, 533, 535, 584, 599, 600, 613, 615, 635, 640, 642, 676, 679, 683, 685, 690, 691, 699, 703, 706, 712, 730, 736
binding, 36, 142, 158, 160, 361, 363, 368, 554, 572, 577, 580, 597, 642, 671, 685, 698, 734
catalytic, 58, 116, 547, 572, 577, 586, 587, 591, 592, 595, 651, 687, 691, 705
equivalent and independent, 38 independent and non-equivalent, 41 Site-directed mutagenesis, 331, 333, 425,
434, 484, 487, 489, 496, 517, 530, 534, 687, 688, 706
Sodium borohydride, 289, 290, 307, 405, 527
Specificity, 343–346, 479, 486, 501, 526, 527, 532, 534, 651, 676, 715
INDEX XI
Spectrophotometry, 163, 180, 307, 310, 314, 459, 498
absorbance, 194 Squatting, 601–604 State activated, 98, 99 conformational, 270 final, 20, 99 initial, 20, 99 ionisation, 269 pre-steady, 9, 193, 202, 224, 716, 761 steady, 8, 53, 63, 72, 76, 78, 109, 193, 202,
662, 664, 666, 667, 669, 670, 713, 716, 749
transition, 58, 367, 373, 377, 380, 383, 425–427, 431–433, 436–438, 444, 459, 461, 475, 488, 518, 571, 765
Steady states, 381, 385 multiple stable, 76–78 Stereospecificity, 347 Structure, 453, 456, 463, 472, 473, 481, 494,
503, 521, 523, 576, 580, 587, 588, 597, 622, 624, 703
β, 471, 638, 639, 671, 676, 691, 699, 703, 712, 764, 766
dissipative, 248, 759 quaternary, 529, 586, 587 secondary, 681 tertiary (three-dimensional), 229, 236, 247,
251, 452, 467, 480, 491, 497, 502, 507, 513, 514, 520, 522, 528, 578, 581, 590, 614–616, 625, 634, 637, 642, 658, 660, 671, 681, 685, 687, 691, 696, 703, 704, 763, 764
Substrate, 685, 730, 752 suicide, 299, 300 Substrate-binding sub-sites, 459, 460, 640 Subtilisin, 153, 297, 452, 461, 629, 634 Sulfonylation, 306 Systems cascade, 653, 664–666 cyclic, 666 bicyclic, 654, 667, 668 monocyclic, 654, 661, 665 polycyclic, 654, 669 irreversible, 654 blood clotting, 654, 655, 660 complement, 653, 658 closed, 63–65 irreversible, 63, 654 K and V, 554–560 open, 63–65
T Tartrate, 745 Tetranitromethane, 310 Theorem connectivity, 750, 751, 753 summation, 750, 751 Theory of absolute rates, 98 collision, 98 of metabolic pathway control,
749–755 of MICHAELIS and MENTEN, 105 “orbital steering”, 418 Thermodynamics, 5, 15, 175, 237, 238,
262, 552, 568, 569, 606, 637, 763
laws of first law, 15 second law, 16 third law, 18 linear, of irreversible phenomena, 70 statistics, 19 Thermolysin, 478 Threonine, 258, 314, 334, 378, 410, 642, 647,
648, 671 Thrombin, 637, 638, 658 Thrombomodulin, 658 Thymidylate synthase, 729 Titration of active sites, 162, 164, 206 Topoisomerase, 647 Tosyl lysine chloromethyl ketone (TLCK),
292 phenylalanine chloromethyl ketone
(TPCK), 292 Transaldolase, 532 Transaminase, 289, 360, 388, 410 Transcription, 251, 259, 727 Transfer, 478 charge, 284 electron, 238, 405, 407, 507, 512 hydride, 405, 406, 507 proton, 478, 514, 515, 518, 525 Transition allosteric, 559, 589 conformational, 591 of quaternary structure, 587 of tertiary structure, 590 Translation, 251, 259 Triose phosphate isomerase (TIM), 410,
413, 434, 443, 513–518, 765
MOLECULAR AND CELLULAR ENZYMOLOGY XII
tRNA synthetases, 403, 489 amino-acyl, 369, 403, 489, 730 tyrosyl, 434–436, 438, 489 Trypsin, 141–143, 150, 151, 163, 218, 219,
271–273, 292, 297, 344, 345, 363, 429–431, 452, 455, 461, 629, 634–637, 658, 660
Trypsinogen, 452, 453, 455 Tryptophan, 244, 310, 321, 361, 459, 460,
493, 625, 649, 651, 680, 684, 694 synthase, 683, 687, 691, 693, 694 Tyrosine, 259, 310, 381, 479, 484–486, 509,
530, 597, 621, 642, 649, 651 Tyrosyl adenylate, 434, 436, 437, 438 U Ultracentrifugation, 56 Ultrafiltration, 56 Uracil, 738 Urea, 366, 727 Urease, 366, 412, 434 Uricase, 745 Uridylation, 649, 653 Uridyl transferase, 653 UTP, 592–595, 652, 673, 738
V Valine, 295, 368, 644 Variable extensive, 18 intensive, 18 W Work, 25 chemical, of biosynthesis, 30, 33 electrical, 25, 34, 43 mechanical, 34 osmotic, 34 X Xanthine, 745 Z Zymogen, 252, 453, 629, 656, 659 activation, 252, 256, 453, 454, 480, 481 chymotrypsinogen, 255, 452–455, 457 procarboxypeptidase, 480 trypsinogen, 452, 455, 457
INDEX XIII
INDEX
A Abzyme, 439, 440, 536 Acetoacetate decarboxylase, 289 Acetoacetyl-ACP, 708 Acetyl-ACP, 708 Acetylation, 260 Acetylcholinesterase, 288, 293, 344, 362,
366, 412, 413, 434, 439, 447 Acetyl-CoA, 239, 666, 667, 700, 702, 706 Acetyl glucosamine, 648 Acid aspartic, 231, 312 glutamic, 231, 312, 646, 656 ketonic, 519 lipoic, 701, 705 muramic (NAM), 497 Actin, 736 Activation, 97, 137, 139–141, 254, 441, 453,
454, 472, 480, 501, 547, 573, 578, 602, 617, 621, 629, 676
allosteric, 556 of complement, 253, 254 energy, 97, 367, 394, 426, 427, 443 partial, 139 by the substrate, 141, 446 total, 137 volume, 222 Activator, 122, 136, 137, 147, 468, 496, 549,
550, 552, 558, 560, 579, 584, 672, 707
Active site (centre) of enzymes, 11, 103, 264, 267, 270, 280, 331, 373, 416, 457, 460, 461, 469, 471, 472, 481, 482, 484, 496, 498, 506, 511, 524, 530, 531, 533, 536
Activity, 21, 233, 234, 245, 267–271, 323, 324, 327, 328, 561, 612, 641, 653, 717
coefficient, 21 molar, 107 Acylation, 150, 151, 273, 278, 302, 308, 310,
314, 415, 447, 462
Acyl carrier protein (ACP), 1, 707–709 Acyl-enzyme, 149, 152, 278, 279, 361, 420,
447, 448, 458, 459, 463–465 Adenine, 233, 246, 435, 505 Adenosine deaminase, 293, 434, 439 Adenylate, 246, 435–438 cyclase, 261, 620, 621 kinase, 760 transferase, 652, 653 Adenylation, 649, 652, 653 ADP, 54, 260, 580, 582, 622, 644, 662, 666,
734, 736 polyribosylation, 260 ribosylation, 260, 261, 641, 644, 647 ADP-Mg, 182 ADP-ribose, 260, 645 Adrenaline, 621 Affinity, 20, 57, 64, 68–70, 112, 137, 139,
141, 149, 553, 554, 556, 557, 559, 562, 569, 584, 587, 592
chemical, 68–70 Alanine, 231, 341, 368, 479, 489, 671, 703 Alcohol dehydrogenase, 166, 168, 501–507,
715 Aldehyde, 507 Aldimine, 521, 522, 524, 525 Aldolase, 526–534, 712, 713, 736 class I, 527 class II, 527 KDPG, 527, 528, 531 Aldose-ketose isomerases, 409, 410 Alkylation, 302, 307, 308, 310, 314 Allosteric effector, 549, 559, 572, 597, 599, 646, 652,
653, 662, 666, 667, 736, 754 model concerted, of MONOD, WYMAN,
CHANGEUX, 553–560, 578 generalised, 567 sequential, of KOSHLAND, NEMETHY,
FILMER, 561–566 Allostery, 548, 572, 698, 707 Amido transferase, 695
Amino acids, 232, 242, 244, 281, 284, 302, 331, 332, 518–520, 526, 531
hydrophobic, 295, 528 Amino-acyl adenylate, 489 AMP, 233, 298, 402, 403, 573, 574, 578, 579,
582, 584, 585, 619, 644 cyclic (cAMP), 77, 259, 298, 620–623,
647, 667, 760 Amylase, 234, 260 Analysis, 323 statistical data, 61, 120, 183 Anthranilate, 680, 685, 686 synthase, 334 Anti-cooperativity, 548, 563, 564, 606–609,
745–747 Apoptosis, 673, 674, 676 Apparatus continuous-flow, 202, 203 GOLGI, 727, 729 quenched-flow, 205 stopped-flow, 204, 205, 224, 225 Approximation of quasi-equilibrium, 112, 132, 137, 175 of the steady state, 174 Archaebacteria (Archae), 241, 671 Arginine, 151, 260, 320, 642, 646, 737 Arylation, 302, 307, 308, 310, 314 Asparagine, 260, 480, 583, 647 Aspartate, 194, 313, 362, 463, 585–587,
591, 593 amino transferase, 179–181, 270, 272, 289,
518–526 2-oxoglutarate amino transferase, 179,
289 transcarbamylase (ATCase), 194, 220, 362,
365, 433, 585–595, 695, 737 Aspartokinase, 602 Aspirin (hydrolysis of), 414, 424 Association, 733–736 enzyme-substrate, 267, 268, 272, 348, 360,
365, 369, 370, 374, 474, 485 ATP, 29, 201, 202, 287, 366, 402, 434–437,
573, 579, 580, 582, 584, 585, 592–595, 599, 600, 619–621, 641, 662, 667, 672, 697, 734–737
hydrolysis of, 29–32 ATPase, 298, 625, 673 ATP-Mg, 182, 183, 490, 491–493, 496, 611,
612, 622, 697 B Binding, 1, 2, 3 complement, 659
ligand, 51, 282 polynome, 50 Biosynthesis, 649, 650, 695, 738, 754 Biotin, 411, 699, 707 Blood clotting, 253, 259, 629, 637, 654, 655,
659 Bond covalent, 284, 360 π, 360 glycosidic, 232, 258, 279, 644 hydrogen, 282, 358, 359, 577, 643, 644,
676, 684, 698, 711 peptide, 232, 242, 282, 632, 656, 658 Butyryl-ACP, 708 C C6-acyl and C16-acyl-ACP, 709 Calmodulin, 623–625 Carbamyl aspartate, 194 phosphate, 194, 585, 591, 697–699, 737 synthetase (CPSase), 695, 697, 700, 737 Carbamylation, 194, 293, 303, 585, 586 Carbanion, 379, 388, 405, 407, 408, 410, 512,
532, 701 Carbene, 295, 296, 298 Carbocation, 379, 405, 431, 499 Carbodiimide, 312, 313 Carbonic anhydrase, 412, 413 Carboxylation of glutamyl residues, 259 γ-carboxyglutamyl, 259 Carboxypeptidase, 361, 362, 366, 453,
478–489, 641 CARNOT’s principle, 63 Caspase, 673–677 Catalase, 344 Catalysis, 101, 280, 335, 384 chemical, 377, 378 electrophilic, 387 enzymatic, 377, 432 general acid, 389, 390, 392, 477, 499, 512,
676 general acid-base, 391, 392, 478, 512,
517 general base, 385, 392, 445, 486 heterogenous, 101 homogenous, 101 intermolecular, 413–416 intramolecular, 413–415 monofunctional, 424 nucleophilic, 380, 384 polyfunctional, 389, 423, 424 Catalyst, 4, 100, 101, 377, 387, 390, 515, 516
772 MOLECULAR AND CELLULAR ENZYMOLOGY
Catalytic efficiency, 343, 362, 414, 427, 433, 448 triad, 363, 457, 462, 470, 616 Cell, 725, 727–732, 735 Cellular enzymology, 725 Cell wall, 733, 736 plant, 736, 747 growth of, 748 Channelling, 679, 681, 687, 691, 694, 697,
699, 705, 712, 717, 718 Chemical kinetics, 85 first order, 88, 89 pseudo-first order, 92 second order, 93, 94 zero order, 96 Chemical labelling, 280 affinity, 290–295 by a coenzyme, 286–290 differential, 301 direct of amino-acid carboxylates (aspartate
and glutamate), 312–313, 314 of α- and ε-amino groups, 302–308 of arginine, 320 of carboxyl groups, 312–313 of histidine, 308, 309 of hydroxyl groups (serine and
threonine), 314–318 of methionine, 319 of sulfhydril groups, 314–318 of tryptophan, 321 of tyrosine, 310, 311 photoaffinity, 295–298 principle of, 280 by a quasi-substrate, 286–290 by suicide reactants, 299 Chemical modifications (post-translational),
257–261 Chirality of biological molecules, 247 Chloramphenicol acetyl transferase, 704 Chorismate, 680 Chromatography, 55 Chymotrypsin, 153, 255, 256, 286, 288, 291,
292, 299, 345, 366, 388, 420, 428, 429, 447, 452–465
Chymotrypsinogen, 255, 456–457 Citrate, 707 synthase, 718 CLELAND nomenclature of, 167, 184 rules of, 177 Coefficient control, 750 elasticity, 750, 751
electrostatic interaction, 43 phenomenological, 71, 74 of transmission, 99 Coenzyme, 166, 387, 388, 406, 407, 519,
522, 700, 715 A (CoA), 239, 241, 705 Colipase, 613, 614, 618 Compartmentalisation, 730, 738 cellular, 727, 730 metabolic, 737, 738 Complex, 335, 633, 635, 637, 638, 659, 661,
670–673, 730 enzyme-activator, 137 enzyme-inhibitor, 132, 498 enzyme-substrate, 105, 127, 132, 137, 155,
171, 206, 335, 347, 361, 365, 369, 420, 441
ES, 137, 154, 422, 424, 431 intermediate, 6, 347, 431 lipase-colipase, 613 of lysozyme with diverse inhibitors, 498 MICHAELIS, 150, 274, 276, 278, 279, 348,
360, 361, 365, 407, 413, 422, 424, 426, 444, 448, 458, 459, 461, 525
multi-enzyme, 679, 700, 702, 707, 717, 737, 749
tetrahedral, 381, 461, 477 Concentration, 731, 752 reduced local-substrate, 743, 744 reduced substrate, 548, 555, 743 Conservation of energy, 65 of mass, 48, 54, 64, 68 Constant, 556 affinity, 553, 562, 633 allosteric, 222, 553, 557, 601 BOLTZMANN’s, 43, 75, 99 catalytic (kcat), 112, 139, 141, 155, 159,
430, 435, 474, 489, 554, 556, 559, 570, 583, 633, 662, 686
dielectric, 21, 43, 273, 280, 349, 441, 444 dissociation, 111, 112, 130, 132, 137, 141,
154, 158, 169, 171, 196, 367, 382, 389, 391, 395, 434, 438, 554, 555, 559, 570, 618, 624, 744
equilibrium, 21, 23, 26, 27, 73, 209, 221, 400, 408, 433, 606, 715
inhibition, 130, 361, 366, 460 intrinsic association or microscopic, 38, 40,
44, 50 ionisation, 274, 277, 278 kinetic, 151, 181 MICHAELIS (Km), 106, 112, 123, 127, 130,
134, 138, 149, 154, 155, 166, 169,
INDEX 773
172, 196, 198, 269, 275–279, 361, 400, 459, 488, 717
PLANCK’s, 99, 356 specific rate, 7, 164, 207, 211, 212, 222,
279, 290, 301, 323, 327, 382, 400, 433, 446, 512, 694
Constraints, 373 diffusional, 745 electrostatic, 745 Control, 749 of linear metabolic pathways, 749–755 of metabolic cycle, 755–761 Cooperativity, 44, 46, 248, 548, 550, 552,
559, 571, 573, 585, 600, 606, 607, 608–611, 745–747
Coupling, 248 diffusion-reaction, 741, 743–745 energetic, 27, 30 fundamental equation, 741 thermodynamic, 70, 567, 569 Crotonyl-ACP, 708 Cryoenzymology, 220 Cyanogen bromide, 319 Cycle cellular, 676, 739 futile, 755 KREBS, 82, 518, 727, 731, 737, 754 limit, 758 metabolic, 755 pentose, 754 tricarboxylic, 22 urea, 518 Cysteine, 244, 281, 314–318, 380, 468, 470,
471, 586, 642, 691, 694, 710 Cystine, 318 Cytidine deaminase, 412, 432, 433 Cytochrome, 508, 509 c, 512, 513, 675 Cytoplasm, 646, 731–733, 739 Cytoskeleton, 624, 670, 676, 731, 735, 736 Cytosol, 670, 706, 712, 729 D DATP, 599, 600 DCTP, 599, 739 Deacylation, 151, 152, 154, 156–158, 278, 415,
416, 420, 444, 447, 458, 463, 465 Deamination, 308 Decarboxylase, 412 Dehydrogenase, 81, 364, 372, 378, 406, 491,
501 alcohol, 501–507 glucose-6-phosphate, 148, 183, 201, 330
α-ketoglutarate, 706 lactate, 366, 445, 502, 534, 535 malate, 502 pyruvate, 666, 700, 702, 704 Denaturation, 24, 269–271 irreversible, 269, 270 reversible, 270 Desoxyribonuclease, 632 Desoxyribonucleotides, 596, 599, 600 Dialysis dynamic, 52, 53, 584 equilibrium, 51, 179 Diffraction neutrons, 463, 534 X-rays, 10, 268, 463, 478, 534 Diffusion, 115, 399, 743–745 rate, 514 substrate, 115, 741 Dihydrofolate reductase, 718 Dihydrolipoamide acetyl transferase, 700 dehydrogenase, 700 Dihydroorotase (DHO), 695 Dihydroxyacetone phosphate, 513, 514, 516, 518 Diisopropyl phosphofluoridate (DFP), 288 Dimerisation, 216 1,3-diphosphoglycerate, 490, 496 Dipole, 341, 348, 350, 352, 357, 378, 441 induced, 353, 354, 363, 364 oscillating, 355 permanent, 351, 355 Disulphide bridge, 257, 464, 467, 484, 614,
635, 636, 660 DNA, 1, 244, 245, 249, 251, 331–333, 727,
729–733, 739 ligase, 729 polymerase, 332, 729 Domain (structural), 344, 491, 493–495, 502,
507, 509, 510, 586, 590, 681, 685, 691, 698, 699, 711, 734
catalytic, 502, 504, 658, 703, 704 coenzyme binding, 502, 503, 508 DONNAN effect, 51 equilibrium, 52 DTTP, 739 Dynamics, 734, 764 molecular, 446, 447, 448, 478, 495 structural, 764 E EADIE plot, 60, 116, 117, 123, 124, 126–129,
133–135, 143
774 MOLECULAR AND CELLULAR ENZYMOLOGY
Effects cooperative, 45, 46, 547, 550, 585, 587,
591 diffusional, 730 electrostatic, 283, 284, 441, 443, 745 entropic, 421 heterotropic, 554, 556, 585, 592, 593 homotropic, 554, 556, 592 isotope, 393–401, 478, 507, 514, 515, 517,
534 magnitude of, 399 primary, 393, 394 secondary, 396, 397, 398 with tritium, 395 orientation, 416, 418, 419 solvent, 395, 396, 397 steric, 283, 284, 398 Elastase, 150, 428, 452, 453, 458, 629, 632,
634, 637, 638, 658 Electron, 510, 512, 538 microscopy, 618, 710 Electrophile, 377, 379, 380, 387, 477, 507,
519 Endergonic, 20, 27, 33, 80 Endopeptidases, 465 Energetics, 1 of enzyme-substrate associations, 365 Energy, 349, 358, 359, 366–368, 384, 385,
395, 425–428, 447, 501, 518 activation, 97, 101, 367, 426–428, 570,
573, 606, 610 association, 426, 489 barrier, 98, 99, 377, 380, 425 binding, 499 change, 20, 24, 25 free, 19, 20, 23, 46, 49, 69, 365, 552, 568,
569 interaction, 349, 354, 369, 551, 569 enzyme-substrate, 46, 49 internal system, 16 potential, 350–353, 355–357 standard free, 20, 21 Enolase, 713 Enoyl-ACP hydratase, 708 reductase, 708 Enthalpy, 277, 365, 366, 369 change, 16, 20, 22–24, 66, 100, 209, 217 Entropy, 15–20, 33, 66–70, 99, 237, 366, 370,
413, 419–422 Enzyme allosteric, 547, 549, 572, 749, 753, 759 angiotensin-converting, 478 condensation, 708, 710
immobilised, 741, 745, 748 kinetics of reactions catalysed by, 743 interconversion, 653, 654, 662, 663, 667,
670 membrane, 730, 733 mnemonic, 602, 605–611 oligomeric, 532, 555, 561 plant cell wall, 736, 747, 748 Equation ADAIR, 48, 552, 567 HILL, 47, 550, 551 LINDERSTRØM-LANG, 44 MICHAELIS, 106, 111, 115, 150, 184, 186,
188 integrated, 118, 143, 196 Equilibrium, 105, 207, 360, 553–554, 562,
568, 611, 759 conformational, 209, 271, 554, 557, 563 far from, 63, 64, 74 protein-ligand association, 37 thermodynamic, 101, 570 Esterases, 153, 439, 447 Eubacteria, 241 Eukaryotes, 241, 670, 671, 672, 729 Evolution, 2, 229, 230, 241, 242, 262, 333,
452, 601, 671, 673, 697, 712, 761 protein, 2 species, 2 time-scale, 229, 230 Exchange hydrogen/deuterium, 764 isotopic, 515 Exergonic, 20, 29, 365, 369, 374 Exon, 235, 245, 503 Exopeptidases, 670 F Factor blood clotting, 253, 259 probability, 38 signal amplification, 664 statistical, 46 tissue, 655, 656 VON WILLEBRAND, 655 FAD-FADH2, 27, 81 Fatty acid synthase, 1 Fibrin, 253, 656, 658 Fibrinogen, 253, 656, 659 FICK’s first law, 742 second law, 742 Flavin, 405, 406, 501, 507–510, 512 Flavocytochrome b2, 407, 501, 507–513
INDEX 775
Flow methods continuous, 202 quenched-flow, 205 stopped-flow, 204 Fluctuations, 79 local, 70 Flux, 71, 72, 666, 730, 750, 751, 753, 755 ATP, 666 matter, 72 metabolic, 73, 82, 679, 715, 719, 749 substrate, 73 FMN, 81, 507, 709 Focus stable, 78, 758 unstable, 78, 758 Force, 348 electrostatic interaction, 348–353, 357 induction, 353, 357 LONDON’s dispersion, 348, 355 Fructose-1, 6-diphosphate, 526, 527, 579 Fructose-6-phosphate, 734 Fructose bisphosphate, 736 Fumarase, 141 Function catalytic, 3 linked, 48 motility, 3 photoreceptor, 3 regulatory, 550, 554 saturation, 41, 50, 106, 107, 555, 563 of state, 554 transport, 3 G α-galactosidase, 345 β-galactosidase, 140, 141, 153, 161, 162, 165,
195, 206, 221, 295, 345, 366, 429, 432
Gene, 503 fusion, 502, 685, 696 GIBBS (free energy), 262 Glucagons, 621 Glucanase, 748 Glucokinase, 201 Glucose, 30, 80, 182, 404, 573, 611, 612,
734, 737, 754 Glucose-1-phosphate, 575, 648, 754 Glucose-6-phosphate, 181, 183, 201, 287,
330, 404, 573, 578, 611, 644, 754, 755
Glutamate, 361, 363, 378, 680, 691, 738 Glutamine, 334, 680, 695, 697, 699, 705 synthetase, 649, 653, 669
Glutarate, 180, 181 Glutaredoxin, 596 Glutathione, 596 Glyceraldehyde-3-phosphate, 334, 364, 490,
513, 515, 516 dehydrogenase, 297, 364, 380, 502, 566,
572, 712–715, 733, 734, 736 Glycerolphosphate dehydrogenase, 713 Glycine, 231, 361, 394, 460, 479, 673 Glycogen, 573, 642, 730, 754, 755 phosphorylase, 220, 234, 259, 417,
573–578, 602, 642–644, 669 See also Phosphorylase a and b synthetase, 642 Glycolate oxidase, 509, 510 Glycolysis, 727, 730, 759, 761 Glycoproteins, 465, 632, 633, 656, 657 Glycosidases, 431, 432, 447, 496, 501 Glycosyl enzyme, 499, 501 groups, 404 Glycosylation (N- and O-), 258, 647–649 β-glycosyl transferase, 404, 602, 648 Graphical plot ARRHENIUS, 100 DIXON, 124, 126, 133–135 EADIE, 60, 124, 566 EISENTHAL and CORNISH-BOWDEN, 118, 119 HANES-DIXON, 118 HILL, 48, 551 from integrated rate equation, 118, 131 KLOTZ, 40, 60 LINEWEAVER-BURK, 60, 124, 566, 606, 609,
612 SCATCHARD, 40–42, 46, 60 semi-logarithmic, 116 VAN T’HOFF, 100 Graphs (method of), 145–148 GTP, 245, 673 H Haem, 238, 508, 509, 512, 559 Haemoglobin, 10, 203, 220, 264, 550, 552,
553, 734, 763 HALDANE, 109, 170 relation, 108 Heat (CLAUSIUS’ non-compensated), 69 α helix, 446, 452, 467, 480, 491, 513, 528,
577, 597, 598, 622, 624, 625 Heparin, 657, 658 Hexose, 247 High pressure, 223, 224 enzyme reactions under, 221
776 MOLECULAR AND CELLULAR ENZYMOLOGY
HILL, 551 coefficient (number), 47 diagram, 47 equation, 47, 550 number, 47, 606 Histidine, 222, 244, 280–282, 297, 307–309,
363, 378, 380, 456, 458, 463, 470, 471, 479, 484, 493, 509, 515–517, 522, 642, 704
Homeomorphic reaction schemes, 177 Homeostasis, 725 Hormone, 253, 620, 632, 637, 647 adrenocorticotropic, 254 β-endorphin, 254 enkephalin, 254 lipotropic (β-LPH), 254 melanocyte-stimulating, 254 Hydrolysis, 132, 141, 142, 152, 161, 165,
166, 233, 245, 273, 303, 316, 319, 363, 389, 401, 414, 420, 424, 428–431, 496, 666
acid, 315 enzyme, 166 Hydrophobic, 272, 445, 476, 479, 485, 492,
509, 529, 530, 532, 594, 616, 621, 635, 639, 660, 684, 699, 712, 733, 734
interaction, 263, 359, 363 Hydroxylation of lysines, 258 of prolines, 258 Hysteresis loop, 743 I Imidazole, 222, 224, 233, 280, 308, 309, 378,
380, 415, 416 IMP, 573 Inactivation, 285, 322, 650 irreversible, 670, 691 Indole, 334, 687, 690, 695, 697 Indole-3-glycerolphosphate (IGP), 680 synthase, 685, 686 Indole propanolphosphate, 691 Induced fit, 370–373, 422, 423, 561 Induction force, 357 Inhibition, 122, 124, 125–136, 273, 299, 300,
361, 480, 584, 602, 641, 734, 744, 747
allosteric, 556, 557 competitive, 122, 124, 549, 736, 745 non-competitive, 125, 126 partial, 132 competitive, 133
mixed, 135 non-competitive, 134 by the reaction products, 130, 131 by the substrate, 129 total, 122 uncompetitive, 127, 128 Inhibitors, 122, 124–127, 147, 172, 177, 178,
218, 256, 273, 288, 290, 322, 361, 362, 366, 371, 431, 432, 523, 549, 550, 556, 557, 559, 584, 632, 641, 661, 672, 684, 691, 698, 744, 745, 752
allosteric, 560, 589, 734 dead-end, 177, 178 protease, 632, 637, 641 serine protease, 288, 632 bovine pancreatic (BPTI), 461, 633, 634 hirudine, 638, 639 irreversible, 122 serpins, 633, 637, 638, 658, 673 antiplasmin, 637 α1-antiprotease, 632, 637 ovalbumin, 637 plakalbumin, 637 soybean, 634 of thiol proteases, 639, 640 bromelain, 640 cathepsin B1, 640 cystatin, 640 stefin, 640 Insulin, 10, 254 Interactions, 55, 348–357, 492, 511, 562, 563,
589, 612, 623, 625, 731, 733 electrokinetic, 348, 355, 357 electrostatic, 42, 43, 272, 284, 348, 357,
358, 495, 518, 526, 734, 746 of enzymes with cell components, 733 hydrophobic, 1, 644 induction, 353 KEESOM, 353 repulsive, 356 VAN DER WAALS, 348, 358, 362, 368, 461, 643 Intermediate, 381 reaction, 206 tetrahedral, 381, 401, 402, 458, 461, 463,
477, 488, 635, 676 Intron, 245, 503 Iodation of histidine, 308 of tyrosine, 260, 308, 310 Iodoacetamide, 314, 319 Ion, 348, 350, 353, 377, 379 hydride, 507 oxonium (oxyanion), 461
INDEX 777
Ionic strength, 21, 43, 52, 273, 362, 455, 495, 719, 734, 746, 747
Ionisation, 270, 274, 276, 278, 279 Isoenzymes, 521, 527 Isoleucine, 270, 295, 361, 368, 738 Isomerisation, 214, 217 K Kcat/Km (ratio), 277, 279, 367, 412, 413, 422,
423, 426, 428–430, 434, 436, 440, 633, 687
Ketone, 307 Kinase, 259, 298, 372, 490–496, 666, 729 adenylate, 31, 760 creatine, 166, 490 hexokinase, 181–183, 199, 200, 268, 404,
417, 490 phosphofructokinase, 259, 490, 572,
579–585, 734, 736, 759 phosphoglycerate, 490, 713, 715 protein kinase, 490, 620–623, 676 See also Phosphorylation protein kinase C (PKC), 733 pyruvate, 200, 330, 490, 713, 759 Kinetics, 582, 605, 608, 609 enzyme, 484 KING and ALTMAN, 168, 172, 174 graphical method of, 143, 144 KLOTZ plot, 40 Km, 717 L β-lactamase, 299, 300, 334, 413, 765 Lactate, 502, 534, 535 Leucine, 361 LINEWEAVER-BURK plot, 60, 117, 120, 123, 124,
126–129, 133–135, 566, 606, 609 Lipase, 613–619 Lipoamide, 702, 703, 705 Localisation (cellular), 642 Lysine, 151, 258, 272, 289, 290, 305, 307, 330,
460, 472, 484, 520–522, 526, 531, 642, 646, 673, 684, 688, 701, 705, 712
Lysosomes, 727, 729 Lysozyme, 10, 372, 373, 425, 431, 481,
496–500, 763, 765 M Malate, 535 Malonyl-CoA, 707 ACP transacylase, 711
Mannose, 648, 661 Mass action (law of), 7, 27, 86, 89, 91, 93 spectrometry, 501 Mechanism, 375, 436, 438, 462, 507, 511,
512, 514 Bi Bi, 166 iso-ordered, 167 ordered, 167, 168, 170, 171 ping-pong, 168, 172, 173 random, 168, 174–176 catalytic, 364, 377, 459, 471, 477, 479,
486, 487, 495, 496, 499, 506, 524, 532, 671
ping-pong, 168, 172, 173 random Bi Uni, 138 Ter Bi, 166 Ter Quad, 166 THEORELL-CHANCE, 168 Uni Bi, 166 Membrane, 34, 235, 236, 646, 647, 655,
659–661, 710, 728, 730, 731, 733, 734, 736, 737, 741, 746
plasma, 727 Metabolism, 343, 412, 662 cellular, 240, 519, 545, 649, 727, 733 theory of, 749, 755 regulation of, 136 semi-cellular, 241 at a surface, 236 Metabolites, 727, 751 compartmentalisation of, 737, 738 flux of, 749, 750, 754 Methionine, 244, 252, 280, 282, 285, 290,
294, 314, 319, 479, 685 Methods chemical, 534 for studying enzyme reactions, 193 continuous, 194 discontinuous, 194 flow, 202–205 potentiometric, 195 relaxation, 206 chemical, 207 spectrum of, 211 thermal (T-jump), 209 MICHAELIS’ law, 233, 743 Microenvironment, 441 Microtubules, 736 Mitochondria, 712, 727, 729, 731 Molecular chaperones, 263 modelling, 286, 333, 369, 425, 434 Muscular contraction, 641
778 MOLECULAR AND CELLULAR ENZYMOLOGY
N N-acetyl-glucosamine (NAG), 372, 431, 497,
498, 500 NADH-NAD, 238, 239, 363, 364, 378, 406,
490, 491, 501, 504–506, 666, 711–714, 734, 737, 759
NADPH-NADP+, 239, 406, 709 NAM, 239, 431, 497–499 N-ethyl maleimide (NEM), 315, 334 Nicotinamide, 712 Nitrene, 295, 298 Node stable, 758 unstable, 758 N-phosphonacetyl-L-aspartate (PALA), 365 Nuclear magnetic resonance (NMR), 363,
634, 638, 754, 755, 765 Nucleic acids (abiotic formation of), 233 Nucleophile, nucleophilic, 153, 154,
156–162, 280, 281, 284, 288, 302, 306, 307, 377, 379, 380–387, 401, 402, 414, 425, 458, 462, 671, 711
attack, 383, 410, 425, 477, 488, 496, 524 competition, 153, 157, 159–166 Nucleotides, 592, 593, 624, 737, 738 abiotic formation of, 233 purine, 592 pyrimidine, 592, 649 Nucleus (cell), 646, 670, 676, 727, 729,
737 Number AVOGADRO(’s), 354 HILL, 551 O Orbital, 418 Order, 85 global, 86 and molecularity, 97 partial, 86 of a reaction, 86, 87 with respect to concentration, 88 with respect to time, 87 Original soup hypothesis, 231 Ornithine, 698 transcarbamylase (OTCase), 737 Oscillations, 760, 761 amplified, 77, 758 damped, 758 glycolytic, 77, 759 in the membrane potential, 760 periodic, 77, 79
sustained, 760 temporal, 79 Oxaloacetate, 344, 520, 718 Oxidation, 30, 32, 258, 310, 501, 507 2-oxoglutarate, 526 Oxyanion, 461, 471, 477, 635, 676, 698 Oxydoreduction, 258, 404–406, 501 potential, 27, 28 reactions, 81 P Palmitate, 709 Palmitoylation, 261 Papain, 153, 425, 446, 465–470, 639 Parachloro mercuribenzoate (PCMB), 315 Parameters, 169, 440 kinetic, 107–109, 116, 131, 149, 150,
156–158, 166, 170, 268, 275, 278, 427, 429, 430, 437, 536, 685
thermodynamic, 374, 420 Paranitrophenyl acetate, 286, 384, 415, 416 guanido benzoate, 286 Pectin, 748 Pepsin, 366, 425, 428, 471–473, 476 Pepstatin, 475 Peroxidase, 221, 310, 344, 378, 379 pH, 195, 233, 235, 269–279, 361, 386, 389,
392, 425, 462, 472, 476, 484, 506, 591, 633, 686, 716, 736, 746, 748
effect of on the conformational state, 270, 271 on the ionisation state of catalytic
groups, 274 profile, 275, 392 Phase latent, 140 pre-steady state, 104 steady state, 104, 140 Phase plane, 758 Phenylalanine, 361, 469, 479 Phenyl methane sulfonyl fluoride (PMSF),
288, 289 Phosphatase, 259, 380, 641, 654, 666 acid, 380, 747 alkaline, 380, 412, 434, 447, 727 Phosphate, 490, 492, 496, 684, 685, 699, 754 pyridoxal, 179, 180, 238, 289, 290, 331,
387, 388, 519, 520, 523, 577, 642, 687, 692
pyridoxamine, 179, 180, 519–522 Phosphatidylinositol, 734 Phosphatidylserine, 734
INDEX 779
Phosphocreatine, 754 Phosphodiesterase, 245, 646 Phosphoenolpyruvate, 408 Phosphofructokinase, 259, 490, 572,
579–585, 734, 736, 759 Phosphoglucomutase, 287, 713 3-phosphoglyceraldehyde, 32 3-phosphoglycerate, 32, 408, 490, 493–495,
526, 527 Phosphoglycerate kinase, 268, 490–496, 713 Phospholipase, 363, 632, 734 Phospholipids, 655, 656, 733, 734 Phosphopantethein, 707 Phosphoribosylanthranilate, 680, 685 isomerase-indoleglycerol synthetase (PRA
isomerase-IGP synthase), 679, 681, 686
isomerase (PRA isomerase), 680, 685 Phosphoribosyl pyrophosphate, 238, 239 amido-transferase, 699 Phosphorylase a and b, 287, 573–579, 668, 730 Phosphorylated components, 30, 31 thermodynamic scale of, 31 Phosphorylation, 259, 287, 363, 573, 577,
629, 641, 662, 668, 754, 755 oxidative, 82, 727, 737 Phosphoryl-enzyme, 287, 447, 490, 496 Phosphotribose, 237, 242 Photooxidation, 309 Photosynthesis, 80, 81 pK, 146, 222, 271–273, 276–279, 383, 389,
445, 463, 476, 481, 526, 591 apparent, 49 Plot primary, 170, 171, 173, 176 secondary, 171, 173 Polarisability, 354, 356
α0, 354 Potential, chemical, 22, 68 Prebiotic chemistry, 231 Pressure, 210, 221, 223, 224, 691 Procolipase, 614, 615, 617 Prohormone, 252 pro-ACTH, 254 proinsulin, 254 Prokaryotes, 253 Proline, 432, 682, 703 racemase, 408, 432 Protease, 108, 288, 344, 380, 446, 452–489,
633, 634, 658, 676, 696 aspartyl, 471–478 chymosin (rennet), 471 endothiapepsin, 473
gastricin, 471 penicillinopepsin, 471, 473, 475, 476 pepsin, 471, 473 rennin, 471, 472 rhizopuspepsin, 473 cysteine, 153 metalloproteases, 378, 478–489, 641 serine, 149, 151, 153, 166, 221, 254, 271,
286, 288, 331, 380, 415, 425, 427, 452–465, 632, 634, 655
chymotrypsin, 150, 153, 166, 255, 256, 271, 286, 292, 331, 344, 345, 366, 415, 417, 420, 428, 429, 446, 447, 451, 453–465, 629
elastase, 428, 452, 463, 629, 632, 634, 637, 658
α-lytic, 452 subtilisin, 452, 629, 634 trypsin, 150, 151, 163, 166, 218, 219,
271, 286, 292, 331, 345, 363, 429–431, 452, 455, 629, 634, 636, 658, 660
thiol, 166, 446, 465–471, 632, 673 actinidine, 465, 466, 468, 469 bromelain, 465, 466, 468, 469, 640 cathepsin, 465, 466, 468, 469, 640 chymopapain, 465, 466, 468 papain, 446, 465–470, 639 Proteasome, 670 20s, 671, 672 26s, 672 Protein CAD, 679, 695–697 Proteolysis, 128 limited, 251, 254, 472, 613, 629, 633, 654,
656 Prothrombin, 259, 655, 657 Protobiont, 234 Purine, 231, 232, 242 Pyridoxamine, 519, 522 Pyrimidine, 231, 232, 242, 243, 490, 509,
649, 695 Pyrophosphate, 402 thiamine, 238, 388, 404, 444, 700 Pyruvate, 666, 737 decarboxylase, 232, 233, 700 dehydrogenase, 666, 679, 700, 701,
702 kinase, 490, 528, 713, 759 Q Quasi-equilibrium, 7 approximation, 112, 132, 139, 175, 176
780 MOLECULAR AND CELLULAR ENZYMOLOGY
R Racemase, 408, 432 Rate, 730, 749, 751 average, 85 diffusion, 412, 413, 514, 731, 743 initial, 8, 113, 114, 130, 154 maximal, of reaction (Vm), 106, 107, 123,
656 reaction, 115, 116, 556, 611, 743, 752,
753 Reaction, 297, 303, 307, 314–316 bimolecular, 72, 213, 214, 218 coupled, 27 elimination, 408 enzyme, 400, 409, 425 one substrate, 137, 143, 148 two substrate, 132, 166 first order, 88, 89 irreversible, 287 isomerisation, 214 pseudo-first order, 92 rate of, 556, 611 rearrangement, 408 reversible, 90 second-order, 93, 94 transfer, 451 acyl, 402 of glycosyl groups, 404, 405, 451 of phosphoryl groups, 402, 451, 489 zero-order, 96 Reactivity, 160, 382, 441 Regression multilinear, 186 non-linear, 120, 184, 186, 188 simple, 184 Regulation, 251, 259, 261, 547, 618, 623,
629, 641, 642, 644, 647, 655, 657, 661, 667, 670, 677, 715, 725, 736, 749
allosteric, 547, 573, 579, 581, 599, 642, 654
of cell metabolism, 733 covalent, 629, 677 of enzyme activity, 8, 259, 679 metabolic, 136, 573, 730 non-covalent, 626 by phosphorylation, 641, 642 Relaxation thermal, 209–219, 498 times, 213, 217 Replication, 729 Reticulum (endoplasmic), 252, 253, 727, 729
Retroinhibition, 248 Reversibility, 108 microscopic, 177 Ribonuclease, 153, 262 Ribonucleotide reductase, 596–601, 729 Ribonucleotides, 596, 600 Ribose, 492, 505 Ribosomes, 236, 252, 727–730 RNA, 245, 246, 249 mRNA, rRNA, sRNA and tRNA, 248, 368,
369, 434–436, 438, 489 polymerase, 259 S Saddle point, 77, 78 Salt bridge, 256, 271, 455, 456 SCHIFF base, 289, 290, 360, 388, 392, 688 Serine, 254, 256, 280, 286–288, 290, 293,
314, 331, 368, 378, 380, 423, 452, 458, 462, 464, 634, 639, 642, 643, 647, 648, 687, 690, 707
Serpin, 633, 637, 638, 673 β sheet, 597, 613, 614, 622, 637, 638 Signal peptidase, 252 peptide, 252, 727 Site (centre) active, 57, 58, 256, 264, 285, 286, 288,
290, 295, 314, 322, 331, 334, 335, 337, 363, 373, 413, 444, 445, 447, 457, 460, 461, 464, 471, 472, 481, 484, 485, 488, 496, 498, 506, 511, 524, 530, 531, 533, 535, 584, 599, 600, 613, 615, 635, 640, 642, 676, 679, 683, 685, 690, 691, 699, 703, 706, 712, 730, 736
binding, 36, 142, 158, 160, 361, 363, 368, 554, 572, 577, 580, 597, 642, 671, 685, 698, 734
catalytic, 58, 116, 547, 572, 577, 586, 587, 591, 592, 595, 651, 687, 691, 705
equivalent and independent, 38 independent and non-equivalent, 41 Site-directed mutagenesis, 331, 333, 425,
434, 484, 487, 489, 496, 517, 530, 534, 687, 688, 706
Sodium borohydride, 289, 290, 307, 405, 527
Specificity, 343–346, 479, 486, 501, 526, 527, 532, 534, 651, 676, 715
INDEX 781
Spectrophotometry, 163, 180, 307, 310, 314, 459, 498
absorbance, 194 Squatting, 601–604 State activated, 98, 99 conformational, 270 final, 20, 99 initial, 20, 99 ionisation, 269 pre-steady, 9, 193, 202, 224, 716, 761 steady, 8, 53, 63, 72, 76, 78, 109, 193, 202,
662, 664, 666, 667, 669, 670, 713, 716, 749
transition, 58, 367, 373, 377, 380, 383, 425–427, 431–433, 436–438, 444, 459, 461, 475, 488, 518, 571, 765
Steady states, 381, 385 multiple stable, 76–78 Stereospecificity, 347 Structure, 453, 456, 463, 472, 473, 481, 494,
503, 521, 523, 576, 580, 587, 588, 597, 622, 624, 703
β, 471, 638, 639, 671, 676, 691, 699, 703, 712, 764, 766
dissipative, 248, 759 quaternary, 529, 586, 587 secondary, 681 tertiary (three-dimensional), 229, 236, 247,
251, 452, 467, 480, 491, 497, 502, 507, 513, 514, 520, 522, 528, 578, 581, 590, 614–616, 625, 634, 637, 642, 658, 660, 671, 681, 685, 687, 691, 696, 703, 704, 763, 764
Substrate, 685, 730, 752 suicide, 299, 300 Substrate-binding sub-sites, 459, 460, 640 Subtilisin, 153, 297, 452, 461, 629, 634 Sulfonylation, 306 Systems cascade, 653, 664–666 cyclic, 666 bicyclic, 654, 667, 668 monocyclic, 654, 661, 665 polycyclic, 654, 669 irreversible, 654 blood clotting, 654, 655, 660 complement, 653, 658 closed, 63–65 irreversible, 63, 654 K and V, 554–560 open, 63–65
T Tartrate, 745 Tetranitromethane, 310 Theorem connectivity, 750, 751, 753 summation, 750, 751 Theory of absolute rates, 98 collision, 98 of metabolic pathway control,
749–755 of MICHAELIS and MENTEN, 105 “orbital steering”, 418 Thermodynamics, 5, 15, 175, 237, 238,
262, 552, 568, 569, 606, 637, 763
laws of first law, 15 second law, 16 third law, 18 linear, of irreversible phenomena, 70 statistics, 19 Thermolysin, 478 Threonine, 258, 314, 334, 378, 410, 642, 647,
648, 671 Thrombin, 637, 638, 658 Thrombomodulin, 658 Thymidylate synthase, 729 Titration of active sites, 162, 164, 206 Topoisomerase, 647 Tosyl lysine chloromethyl ketone (TLCK),
292 phenylalanine chloromethyl ketone
(TPCK), 292 Transaldolase, 532 Transaminase, 289, 360, 388, 410 Transcription, 251, 259, 727 Transfer, 478 charge, 284 electron, 238, 405, 407, 507, 512 hydride, 405, 406, 507 proton, 478, 514, 515, 518, 525 Transition allosteric, 559, 589 conformational, 591 of quaternary structure, 587 of tertiary structure, 590 Translation, 251, 259 Triose phosphate isomerase (TIM), 410,
413, 434, 443, 513–518, 765
782 MOLECULAR AND CELLULAR ENZYMOLOGY
tRNA synthetases, 403, 489 amino-acyl, 369, 403, 489, 730 tyrosyl, 434–436, 438, 489 Trypsin, 141–143, 150, 151, 163, 218, 219,
271–273, 292, 297, 344, 345, 363, 429–431, 452, 455, 461, 629, 634–637, 658, 660
Trypsinogen, 452, 453, 455 Tryptophan, 244, 310, 321, 361, 459, 460,
493, 625, 649, 651, 680, 684, 694 synthase, 683, 687, 691, 693, 694 Tyrosine, 259, 310, 381, 479, 484–486, 509,
530, 597, 621, 642, 649, 651 Tyrosyl adenylate, 434, 436, 437, 438 U Ultracentrifugation, 56 Ultrafiltration, 56 Uracil, 738 Urea, 366, 727 Urease, 366, 412, 434 Uricase, 745 Uridylation, 649, 653 Uridyl transferase, 653 UTP, 592–595, 652, 673, 738
V Valine, 295, 368, 644 Variable extensive, 18 intensive, 18 W Work, 25 chemical, of biosynthesis, 30, 33 electrical, 25, 34, 43 mechanical, 34 osmotic, 34 X Xanthine, 745 Z Zymogen, 252, 453, 629, 656, 659 activation, 252, 256, 453, 454, 480, 481 chymotrypsinogen, 255, 452–455, 457 procarboxypeptidase, 480 trypsinogen, 452, 455, 457
INDEX 783
